Immunoglobulins are glycoproteins, i.e., oligosaccharides are attached to the protein at various sites. Vicinal diols of these oligosaccharides can be oxidized with periodate to yield dialdehydes, and the aldehyde groups thus produced reacted with various amines and hydrazines to form Schiff bases and hydrazones. For example, Willan et al., FEBS Letters, 80 133 (1977) oxidized an oligosaccharide attached to an asparagine residue at position 297 (using the numbering system of the human IgGl myeloma protein Eu) in the C.sub.H 2 region of rabbit IgG. This oxidized material was, after purification, reacted with 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl free radical and the resulting imine (reaction of amino group with sugar aldehyde) reduced with NaBH.sub.3 CN to yield a spin labelled protein. Timofeev et al., FEBS Letters, 89 191 (1978) also used oxidized glycoprotein monoclonal antibodies to prepare spinlabeled material. Murayama et al., Immunochemistry, 15 523 (1978) labeled periodate oxidized oligosaccharidegroups in an immunoglobulin (IgG) with amino-containing compounds via Schiff base formation. Aspartic acid and horseradish peroxidase were the "amines" employed. The aspartic acid-Schiff base was used to detect the antigenicity of human IgG by counter immunoelectrophoresis. et al., Immunology Letters, 8 273 (1984), coupled biotin hydrazide with aldehyde groups of oxidized immunoglobulins and suggested the use of the procedure for conjugation of fluorescent dyes to monoclonal antibodies.
Chua et al., Biochimica and Biophysica Acta, 800 291 (1984), oxidized carbohydrate groups on the constant region of the heavy chain of IgM (which has a relatively high carbohydrate content) to yield aldehydes. These aldehyde groups were reacted with hydrazide groups linked to a liposomal membrane. The monoclonal IgM antibodies were specific to a protein carrying a 1-dimethylamino naphthalene-5-sulfonyl haptan.
Rothfus et al., J.B.C., 238 1402 (1963) used periodate oxidation followed by borohydride reduction to determine the identity of the sugar component of the glycopeptide from human gamma globulin.
The linking of cytotoxic agents to immunoglobulins is reviewed by Blair et al., J. Immun. Meth., 59 129 (1983) and by Ghose, Blair and Kulkarni, Methods in Enzymology, 93 280 (1983). Blair et al. reveal, at page 130, the possibility of linking the cytotoxic agent to the oligosaccharide of an Ig. Oxidation to an aldehyde and coupling of the cytotoxic agent to the aldehyde is not mentioned. Ghose et al. cover much of the same ground, but do mention specifically the periodate oxidation of rabbit anti-BSA IgG having attached oligosaccharides to form aldehyde groups and the reaction of these groups with ethylenediamine followed by borohydride reduction of the Schiff base double bond to form new aminoethyl primary amine groups attached to the oligosaccharide portion of the glycoproteins.
EPO Application publication 88695 broadly claims processes described in the above publications; reaction of an aldehyde derived by oxidation of a carbohydrate moiety with a hydrazine, hydrazide or amine linker group of a compound to form a conjugate. The application is apparently directed to linker groups which are conjugated to the aldehyde-antibody and the linker group itself attached to an insoluble support or a second compound. There are also claims to antibody conjugates comprising any compound attached to a carbohydrate moiety of an antibody via a covalent hydrazone, imine or enamine bond. More specific claims are to a peptide linker, an amino acid linker or a linker of the general formula W(CH.sub.2).sub.n Q where W is C.sub.6 H.sub.5 NH--CH.sub.2 -- or --CH.sub.2 -- and Q is an amino acid, peptide, chelator or chelator derivative. Drugs specifically disclosed for antibodymediated delivery via this system are listed in Table 1 and includes these antineoplastic agents, daunomycin, bleomycin, vinblastine, vincristine, and 5-fluorouracil. There is no teaching of how these drugs are to be linked and, specifically, no teaching of how VLB or vincristine are to be linked since neither contains a hydrazide or amine function. Coupling to Fab antibody fragments is emphasized. Specific disclosure is limited to coupling ALKERAN.RTM. (N-[Bis(.beta.-chloroethyl)phenyl]alanine) and monoclonal antibodies against sheep red blood cells. No in vivo data are provided.
VLB, vincristine, and other antineoplastic drugs have been linked to immunoglobulins or other proteins.
U.K. patent application G.B. No. 2,090,837A discloses immunoglobulin conjugates covalently linked to a vinca moiety by amide formation. The amide bond itself is produced by the reaction of a vinca indole-dihydroindole alkaloid such as 4-desacetyl-VLB via a C-3 carboxazide group with a free amino group of an immunoglobulin or immunoglobulin fragment. The azide is prepared from 4-desacetyl-VLB-3-carboxhydrazide which is itself prepared by the action of hydrazine on VLB. The covalent bond formed between the vinca moiety and the immunoglobulin is not ordinarily reversible; i.e., the bond is not subject to hydrolytic (chemical) cleavage at physiological pH.
U.S. Pat. No. 4,203,898 (Cullinan I) discloses and claims 3-carboxazides of vinca indole-dihydroindole alkaloids such as VLB, vincristine, etc. It is these azides which were used in preparing the covalently linked immunoglobulin conjugates of the previous reference. The azide is prepared from a 4-desacetyl-3-carboxhydrazide of an indole-dihydroindole alkaloid. Such hydrazides are also disclosed and claimed.
U.S. Pat. No. 4,166,810 (Cullinan II) discloses a group of derivatives of 4-desacetyl-VLB-3-carboxhydrazide including N.sup.2 -alkyl derivatives. The compounds are prepared by forming a hydrazone upon reaction of the N.sup.2 amine group with an aldehyde or ketone and then reducing the resulting enamine.
Neuss et al., Tetrahedron Letters, 783 (1968), disclose leurosine hydrazide.
Barnett et al., J. Med. Chem., 21 88 (1978), also disclose the hydrazides of VLB and related indoledihydroindole derivatives.
Conrad et al., ibid, 22 391 (1979), discuss generally the amides of VLB and their activities as anti-cancer drugs. The same amides discussed above in connection with Cullinan I are disclosed herein. In addition, a bridged disulfide, compound 30, was prepared in which the carboxamide group was attached to an ethyldithioethylcarboxamide grouping. The corresponding primary amine containing the disulfide bond is compound 38. Also disclosed are radioimmune assays for VLB and vincristine. The radioimmune assay was developed by coupling 4-desacetyl-vinblastinoic acid azide (4-desacetyl-VLB-3-carboxazide) to BSA. This antigen was used to prepare antibodies in rabbits which antibody would then pick up the VLB moiety of the antigen. The amount of antigen was determined by using VLB labeled with tritium--see also Root et al., F.A.C.S.S., 2nd National Meeting, Oct. 6-10, 1975, Abstract 183.
Hargrove, U.S. Pat. Nos. 3,392,173 and 3,387,001, discloses novel C-4 esters of VLB, vincristine, leurosidine, etc. Among these esters is a chloroacetyl ester, which derivative was employed in EPO No. 124,502 to couple with a protein. Hargrove II reacted this 4-chloroacetyl ester with amines to prepare, for example, an N,N-dimethylglycine ester--vinglycinate.
Langone et al., Anal. Biochem., 95 214 (1979), developed radioimmune assays for vinblastine and vincristine. The antigens used were prepared by oxidizing VLB to a dicarboxylic acid and then coupling this product to a protein with a carbodiimide coupling reagent.
Teale et al., Brit. J. Clin. Pharm., 4 169 (1977), also developed radioimmune assays for vinblastine and vincristine. The vinca alkaloid is conjugated to the albumin by a Mannich reaction using an amine group in the protein (BSA), formaldehyde and vinblastine. The point of attachment of the vinca alkaloid, vinblastine or vincristine, is not specified.
Johnson et al., Brit. J. Can., 44 372 (1981), disclose the preparation of vindesine linked to anti-CEA immunoglobulin via an azide. [It should again be emphasized that the azide from desacetyl VLB and the azide congener of vindesine are identical since the compounds differ only as to the moiety on the C-3 carboxylic acid and this moiety (ester or amide) disappears after the coupling reaction.] These conjugates were found to be cytotoxic for human cancer cells in vitro. U.K. patent application GB No. 2,090,837, inventors Rowland and Simmonds, covers the same subject matter.
Rowland et al., Cancer Immunology and Immunotherapy, 19 1 (1985), discuss the anti-tumor properties of vindesine-monoclonal antibody conjugates (4-desacetylVLB derivatives conjugated via the C-3 carboxazide group as in U.K. patent No. 2,090,837A and Johnson et al). Four conjugates were tested against human tumor xenografts in athymic mice, and all showed some anti-tumor activity. Vindesine was inactive at non-toxic levels with the same tumor.
EPO No. 124502 covers conjugates prepared from an immunoglobulin and a 4-desacetyl-VLB or vindesine or other 4-desacetyl-VLB-3-carboxamide linked via a 4-succinate; i.e., a bridging group or linker between the 4-hydroxy group and the protein. The bridging group of the structure can be CO-CH.sub.2 (from a 4-chloroacetyl group) or CO(CH.sub.2).sub.n --CO (succinic or glutaric acid). The same 4-succinoyl derivatives are disclosed in United Kingdom patent application GB No. 2,137,202, and conjugates formed therefrom are disclosed in GB No. 2,137,210.
Bumol et al., Proc. Am. Assn. Cancer Research, 25 356 Abstract 1410 (1984) (Bumol I), discuss the characteristics of a conjugate prepared according to GB patent No. 2,137,210; i.e., a 4-desacetyl-4-succinoyl-VLB conjugate with a monoclonal antibody to a human adenocarcinoma (KS1/4). Bumol et al., Federation Proceedings, 44 1864 Abstract 8484 (1985) (Bumol II) discuss a similar conjugate with a monoclonal antibody to a human melanoma. Bumol et al., J. Cellular Biochemistry, Supplement 9A Abstract 0124 (1985) (Bumol III), discuss further work on the 4-desacetyl-4-succinoyl-VLB KS1/4 conjugate (adenocarcinoma).
None of the above references disclose or suggest conjugating an amine or hydrazine derivative of an antineoplastic dimeric indole-dihydroindole alkaloid with an oxidized carbohydrate (oxidized to one or more aldehyde groups) on the surface of a monoclonal antibody which is also a glycoprotein.